Springer Lab: TransformationYeast96well

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1. Grow overnight in deep (1.5mL) 96 well plate

2. Dilute between 1:20 and 1:50 (in practice 1:40) and grow for 4 hours

3. Harvest cultures by centrifugation at 3000 rpm for 5 min

3a. combine pellets from multiple plates to increase cell numbers

4. Resuspend in 1mL of sterile water

5. Wash pellet three times in water (spin at 3 000 rpm between washes).

6. Wash pellets three times in 0.1M LiAc

6a. After last wash, keep 35 microL 0.1 M LiAc in each well

7. Prepare transformation mix per well (see table below)

 240 uL PEG (50% w/v)
 35 uL LiAc (1 M)
 10 uL Salmon Sperm Carrier DNA (10mg/mL)
 1 ug transforming DNA (in 30-50 microL)

8.Add transformation mix to each well with multichannel pipette

9. Vortex cells for 1 min

10. Incubate cells at 30C for 30 minutes

11. Heat shock cells at 42C for 25 minutes.

12. Spin cells down at 3 000 rpm for 5 minutes. Remove supernatant with multichannel pipette.

13. Add 600 microL of YPD per well and incubate overnight at 30C

14. Plate cell suspension on appropriate selection media and isolate transformants after 2-3 days

'	1 well	12	24	36	48	60	72	80	84	96
PEG 50%	240	2880	5760	8640	11520	14400	17280	19200	20160	23040
LiAc (1M)	35	420	840	1260	1680	2100	2520	2800	2940	3360
Salmon Sperm DNA (5 mg/mL)	10	120	240	360	480	600	720	800	840	960
transforming DNA	10	120	240	360	480	600	720	800	840	960
Total in microL	295	3540	7080	10620	14160	17700	21240	23600	24780	28320
Total in mL	0.295	3.54	7.08	10.62	14.16	17.7	21.24	23.6	24.78	28.32

For the actual master mix use 10% more reagents	1 well	12	24	36	48	60	72	84	84	96
PEG 50%	264	3168	6336	9504	12672	15840	19008	21120	22176	25344
LiAc (1M)	38.5	462	924	1386	1848	2310	2772	3080	3234	3696
Salmon Sperm DNA (5 mg/mL)	11	132	264	396	528	660	792	880	924	1056
transforming DNA	11	132	264	396	528	660	792	880	924	1056
Total in microL	324.5	3894	7788	11682	15576	19470	23364	25960	27258	31152
Total in mL	0.3245	3.894	7.788	11.682	15.576	19.47	23.364	25.96	27.258	31.152